Fragile X Syndrome (FXS) is the most common single-gene cause of autism and mental impairment, affecting as many as 1 in 2500 children. Children with FXS suffer from a variety of cognitive and behavioral impairments, including hypersensitivity to sensory stimuli. In the well-established Fmr1 knockout (KO) mouse model of FXS, a variety of neuronal defects have been discovered, ranging from abnormalities in synaptic plasticity and dendritic spine stabilization to alterations at the level of cortical circuits. Recet studies have established that neuronal hyperexcitability contributes to circuit dysfunction in Fmr1 KO mice and could lead to sensory hypersensitivity in FXS. Using Fmr1 KO mice, the Portera-Cailliau lab has found that neurons in the barrel cortex, which processes whisker inputs, show abnormally elevated firing during spontaneous activity and high network synchrony during a critical period of early postnatal brain development. It remains unknown whether sensory stimulation might also trigger exaggerated neural responses in Fmr1 KO mice, which could conceivably alter sensory perception. Therefore, the experiments in Aim 1 will test two initial hypotheses: First, in Fmr1 KO mice at both developmental and adult ages, neurons in barrel cortex show excessive firing in response to whisker stimulation, as well as broad tuning (i.e., neurons in a given barrel respond to multiple whiskers) and impaired adaptation to persistent stimulation; and second, the mutant mice have behavioral deficits in sensory processing that impair their decision-making in a whisker discrimination task. Additionally, a growing body of work indicates that dysregulated signaling of the excitatory neurotransmitter acetylcholine (ACh) plays a role in FXS pathophysiology. ACh can modulate attention and the response of neuronal ensembles to sensory input, and there is evidence that the Fmr1 KO mouse exhibits excessive muscarinic ACh receptor (mAChR) signaling, but the actual impact of altered cholinergic tone on the activity of sensory circuits is unknown. Accordingly, the experiments in Aim 2 will also test the hypothesis that an excess of cholinergic tone could account for the pathological hyperexcitability of sensory circuits in the KO mice. Aim 2 will also examine whether pharmacological administration of a cholinergic antagonist can correct the abnormal sensory-evoked responses and rescue the perceptual deficits of Fmr1 KO mice. The proposed research will use cutting-edge in vivo two-photon calcium imaging with the genetically encoded calcium indicator GCaMP6s to record network activity in large ensembles of cortical neurons in barrel cortex, in awake, behaving mice performing a Go/No-go whisker discrimination task. These studies will link, for the first time in a model of a neurodevelopmental disorder, cortical network defects with specific behavioral alterations.